PROJECT 2: Fatty Liver Predisposes to Metastasis: Role of Hepatic Stellate Cells PROJECT SUMMARY The central hypothesis being tested by this Program project is that the normal liver has defense mechanisms to suppress metastatic growth, which are lowered by insults to the liver microenvironment to allow metastatic engraftment and expansion. Project 2 focuses on the role of fatty liver in liver metastasis, examining the hypothesis that non-alcoholic fatty liver disease (NAFLD) associates with a tumor-promoting liver microenvironment that is mediated by hepatic stellate cells (HSCs), hyaluronic acids (HAs), and hepatocyte- derived extracellular vesicles (EVs). Obesity and NAFLD are serious health concerns that increase the risk of primary cancers including liver, pancreas, colon, prostate, and breast. Fatty liver also supports liver metastasis in patients as well as in rodent models. The liver is the most frequent site for metastasis of visceral cancers such as colorectal (CRC) and pancreatic ductal adenocarcinoma (PDAC). The extent of liver metastasis is a major survival determinant for patients with these cancers as well as for those with prostate cancer (PCa). The objective of Project 2, thus, is to investigate molecular mechanism(s) underlying HSC-mediated, enhanced metastatic growth in the fatty liver environment. In liver, HSCs are precursors of liver myofibroblasts that produce extracellular matrix (ECM) components, such as HAs. Both HSCs and the ECM are elemental in supporting a liver tumor microenvironment. This research team's preliminary data show that HSC infiltration and ECM production (as evidenced by HAs) in metastatic tumors were increased in a fatty liver environment. In contrast to previous studies showing that primary tumor- derived EVs travel to the liver to create a pre-metastatic niche, we propose the innovative hypothesis that a pro-metastatic liver microenvironment is instead facilitated by non-cancerous, steatotic hepatocytes that secrete EVs containing pro-fibrogenic and oncogenic microRNAs (miRNAs). This project will examine liver metastasis in fatty liver for three different cancer types (CRC, PDAC, and PCa) via three specific aims. Aim 1 will investigate whether HSC-derived HA promotes metastatic liver tumor growth in fatty liver with HSC-specific deletion or overexpression of HA synthase 2 (Has2), in the presence of high-fat diet (HFD). Aim 2 will investigate whether HA overexpression in fatty liver promotes metastatic tumor growth via CD44, Notch1, and Yes-associated protein (YAP). Aim 3 will investigate whether fatty liver-derived EVs promote cancer metastasis to the liver by activating tumor-promoting pathways via oncogenic miRNAs. We will also seek EV-miRNAs as biomarkers to predict patients at high risk for liver metastasis. This project integrates with others in the Program by examining associations between fatty liver-mediated HAS2 overexpression and YAP (Projects 1, 3) and methionine adenosyltransferase 1A (MAT1A, Project 4).